marine drugs

Article

Two New Alginate Lyases of PL7 and PL6 Familiesfrom Polysaccharide-Degrading BacteriumFormosa algae KMM 3553T: Structure, Properties, andProducts Analysis

Alexey Belik, Artem Silchenko, Olesya Malyarenko, Anton Rasin, Marina Kiseleva,Mikhail Kusaykin * and Svetlana Ermakova

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Laboratory of Enzyme Chemistry,Far-Eastern Branch of the Russian Academy of Sciences, 690022, Vladivostok, 159,Prospect 100-let Vladivostoku, Russia; belik_a_a@mail.ru (A.B.); artem.silchencko@yandex.ru (A.S.);vishchuk@mail.ru (O.M.); abrus__54@mail.ru (A.R.); mikiseleva@mail.ru (M.K.);svetlana_ermakova@hotmail.com (S.E.)* Correspondence: mik@piboc.dvo.ru; Tel.: +7-914-714-04-34

Received: 24 January 2020; Accepted: 23 February 2020; Published: 24 February 2020�����������������

Abstract: A bifunctional alginate lyase (ALFA3) and mannuronate-specific alginate lyase (ALFA4)genes were found in the genome of polysaccharide-degrading marine bacterium Formosa algae KMM3553T. They were classified to PL7 and PL6 polysaccharide lyases families and expressed in E. coli. Therecombinant ALFA3 appeared to be active both on mannuronate- and guluronate-enriched alginates,as well as pure sodium mannuronate. For all substrates, optimum conditions were pH 6.0 and 35 ◦C;Km was 0.12 ± 0.01 mg/mL, and half-inactivation time was 30 min at 42 ◦C. Recombinant ALFA4 wasactive predominately on pure sodium mannuronate, with optimum pH 8.0 and temperature 30 ◦C,Km was 3.01 ± 0.05 mg/mL. It was stable up to 30 ◦C; half-inactivation time was 1 h 40 min at 37◦C. 1H NMR analysis showed that ALFA3 degraded mannuronate and mannuronate-guluronateblocks, while ALFA4 degraded only mannuronate blocks, producing mainly disaccharides. Productsof digestion of pure sodium mannuronate by ALFA3 at 200 µg/mL inhibited anchorage-independentcolony formation of human melanoma cells SK-MEL-5, SK-MEL-28, and RPMI-7951 up to 17%stronger compared to native polymannuronate. This fact supports previous data and suggests thatmannuronate oligosaccharides may be useful for synergic tumor therapy.

Keywords: alginate lyase; enzyme specificity; polysaccharide; oligosaccharide; anticancer activity

1. Introduction

Alginate lyases are among the most studied carbohydrate-modifying enzymes. They are producedby organisms, which use alginic acids as a source of energy, and by alginate-producing bacteria toform exo-polysaccharide (EPS) as the main part of biofilms [1]. Their mechanism of action is based onβ-elimination, i.e., cleavage of a bond between two β-d-mannuronic or α-L-guluronic links with theformation of 4-deoxy-L-erythro-hex-4-enopyranosyluronate (∆) [1].

According to classical nomenclature, alginate lyases are divided into M-specific (EC 4.2.2.3) andG-specific (EC 4.2.2.11), however, there are also lyases with wide specificity that cleave bonds betweenМ-and G- residues. They also have different types of action (exo- or endo-), and alginate oligosaccharidelyases (4.2.2.26) have the exo- type of action. Not all alginate lyases digest acetylated substrate [2], sothe screening for this ability is an important step in the development of new antibiofilm tools. Theoptimum pH for these enzymes ranges from 5 to 10, and the optimum temperature ranges from 20 to

Mar. Drugs 2020, 18, 130; doi:10.3390/md18020130 www.mdpi.com/journal/marinedrugs

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70 ◦C. Km value extends from 0.0026 mM on acetylated alginate (Pseudomonas aeruginosa strain PDO486(DP 263) [3]) to 6.7 mM on polymannuronate from Qingdao BZ Oligo Biotech Co., Ltd. (China) [4].

By the amino acid sequence homology, alginate lyases can be divided into several structuralfamilies of polysaccharide lyases (PL) (http://www.cazy.com): PL5, PL6, PL7, PL14, PL15, PL17 andPL18 [5,6]. Most of the alginate lyases with different specificity belong to PL7, which consists of aβ-jellyroll that forms catalytic cleft and three adjacent β-sheets with catalytic amino acid residues [7].Catalytic domains of PL14 and PL18 also have β-jellyroll fold, while PL6 has a β-helix fold, and PL5,PL15, PL17 have (α/α)n toroid folds [8].

Products of action of alginate lyases reveal multiple biological activities, presumably, due to theirevenly distributed negative charge and ability to strongly bind with a protein molecule, modifying itsconformation. For example, those obtained by Pseudoalteromonas sp. (DP 3–9) stimulate the immunesystem, increasing the production of cytokines (G-CSF, TNF-α, interleukins) in mural macrophages.The most active are unsaturated guluronate G8 and unsaturated mannuronate M7 oligomers [9,10].Products of action of alginate lyase from Agarivorans sp. L11 (only DP 5) inhibit the growth of humanbone cancer cells MG-63 by 60–70% [11]. Products of action of alginate lyase from Sphingobacteriumsp. (DP 2–4) show antioxidative properties in vitro [12]. Alginate oligosaccharides inhibit growth anddifferentiation of adipocytes and absorption of saturated fatty acids [13,14] and exhibit anti-allergicproperties by suppression of IgE [15].

Computer analysis of the distribution of alginate lyases and alginate lyase-encoding genesamong different groups of living organisms reveals that there is still a great potential in experimentalcharacterization of amino acid sequences with potential alginate degrading activity. There are morethan 50 thousand known amino acid sequences, identified as alginate lyases, and 47 thousand ofthem belong to bacterial genomes. Just 260 enzymes have been functionally characterized: 181 fromEC 4.2.2.3, 70 from EC 4.2.2.11 and 9 from EC 4.2.2.26. According to the CAZY database [6], amino acidsequences and functional characteristics are known for 27 alginate lyases.

Thus, the class of alginate lyases is still insufficiently studied, while its practical application isof high importance. Polysaccharide-degrading marine bacterium Formosa algae KMM 3553T (F. algae)is one of the potential sources of genetic material for the expression of alginate lyases; these bacteriainhabit thalli of brown algae Fucus evanescens in the cold waters of Sea of Okhotsk [16].

The strain has been characterized previously as the producer of various carbohydrate-degradingenzymes: fucoidanases FFA1 [17], FFA2 [18], and endo-1,3-β-D-glucanase GFA [19]. Accordingly,F. algae can be described as a psychrotolerant organism, environmentally adapted and evolutionallyspecialized for inhabiting and utilizing brown algae, and containing enzymes, that are active at lowtemperatures. On the other hand, its optimum temperature of growth is 23 ◦C, so there can be otherenzymes, stable at room temperature, that can be used in biotechnology.

In this study, we report on the progress of research on F. algae’s enzymatic machinery forbiotechnology applications and the discovery of new alginate lyases ALFA3 and ALFA4.

2. Results

The yield of the target enzyme was 72 mg/L of cultural media for ALFA3 and 12 mg/L for ALFA4.Molecular weights, structural families of amino acid sequences, and substrate specificity are presentedin Table 1.

Table 1. Properties of recombinant alginate lyases of Formosa algae.

Enzyme Molecular Weight, kDaSpecific Activity, U/mg

EC Number Structure FamilyPolyM G-enriched M-enriched

ALFA3 33.8 21.3 18.7 21.5 4.2.2.3 PL7

ALFA4 89.8 1.3 0.2 2.2 4.2.2.3 PL6

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ALFA3 digested three types of sodium alginates (M-enriched, G-enriched and MG-mixed) withequal effectiveness: 21.5 U/mg for M- and MG- and 18.7 U/mg for G-. The detected difference indicatedthat this enzyme belonged to EC 4.2.2.3 (mannuronate-specific alginate lyase). Optimum pH was 6.0for all types of substrates (Figure 1).

Figure 1. Optimum pH of ALFA3 when acting on different substrates. M – sodium mannuronate,MG – mannuronate-enriched sodium alginate, G – guluronate-enriched sodium alginate. Standarddeviations are given.

The optimum temperature of ALFA3 was 35 ◦C (Supplementary Figure S1), half-inactivationtime was 1 h 30 min at 42 ◦C. Ions of K+ and Ca2+ in concentrations up to 0.5 M did not influence theactivity. Km was 0.12 ± 0.01 mg/mL (Figure S2). ALFA3 Vmax values were 0.128 × 10−3 M/min for G,0.150 × 10−3 M/min for MG, and 0.211 × 10−3 M/min for M. Kcat values were 3.52 s−1 for G, 4.13 s−1

for MG and 5.80 s−1 for M.Alginate lyase ALFA4 had the optimum temperature of 30 ◦C (Supplementary Figure S3), optimum

pH was 8.0, the optimum concentration of NaCl was 0.6 M, half-inactivation time at 37 ◦C was 1 h40 min. Km was 3.01 ± 0.05 mg/mL (Figure S4) of the mannuronate-enriched substrate. Vmax was0.314 × 10-3 M/min for MG. Kcat value for MG was 2.88 s−1.

According to classification based on amino acid sequence, ALFA3 was determined to belong toPL7 polysaccharide lyase family and ALFA4 to PL6 family. As long as ALFA3 and ALFA4 digestedmannuronate-enriched substrate more rapidly than guluronate-enriched, they were classified asmannuronate-specific (EC 4.2.2.3)

Kinetic analysis of substrate digestion for ALFA3revealed that the depth of substrate cleavagestrongly depended on M/G ratio: while sodium polymannuronate was completely digestedto oligosaccharides in 24h (hydrolysis depth 88.3%), guluronate-enriched substrate contained aconsiderable high-molecular fraction (hydrolysis depth 67.9%), while this fraction was much smallerfor the mannuronate-enriched substrate (hydrolysis depth 87.3%) (Figure 2). These fractions are nothydrolyzed even after addition of more enzyme.

Precipitation of alginate poly- and oligosaccharides with ethanol provides from incubation mixtureis a controllable and simple method for isolating low-molecular fraction with a defined range ofalginate oligosaccharides (AOS) (Figure 3). In comparison to anion-exchange chromatography thismethod has no need additional steps of desalting [11]. This diagram shows how to find necessaryconcentrations to obtain the fraction of purpose: first discard heavy fractions (39% of ethanol) ina residue and then precipitate the fraction of purpose with a higher concentration of ethanol (45%of ethanol). Isolating a low-molecular is an important step, because only AOS with low degree ofpolymerization can be potentially used as infusion drugs without a risk precipitation and thickening inblood. Furthermore, exactly AOS have shown the supreme therapeutical potential in compare withhigh-molecular fractions (alginate polysaccharides) [11].

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Figure 2. Kinetic analysis of digestion of different substrates by ALFA3 (A) and final products of digestionof different substrates by ALFA4 (B). M – sodium polymannuronate, MG – mannuronate-enrichedsodium alginate, G – guluronate-enriched sodium alginate. Time of reaction is given in h. Each lanewas loaded with 60 µg of sample.

Figure 3. Products of action ALFA3 on mannuronate-enriched substrate precipitated by ethanol inratios from 8.6 to 45.0% (low-molecular fraction). Numbers correspond to ethanol percentage inprecipitation mixtures. Precipitation was run during 18 h at 4 ◦C.

1H NMR analysis of digestion products for guluronate-enriched substrate (M:G~1:2) showedthat it was successfully hydrolyzed by ALFA3 (Figure 4A, B). The 1H spectrum of the hydrolyzedsample was compared to the previously reported data [20–25] to assign its NMR shifts. Since therewere a visible accumulation of signals for residue ∆G’s H4 (5.87 and 5.95 ppm) and residue ∆M’s H4(5.76 and 5.80 ppm), with the dominant signal from ∆G, the potential ALFA3-hydrolysed bonds wereMM, MG and GM. The disproportionally low intensity of G red signal (5.25 ppm) relative to M red(5.22 ppm) allows concluding that MM, MG and GM but not GG bonds are cleaved. The differences inspectra of high-molecular-weight (HMW) (Figure 4C) and low-molecular-weight (LMW) (Figure 4D)fractions, separated by ethanol precipitation in ratio ethanol: reaction mixture 0.9:1, revealed thatthe HMW fraction contained mainly G-blocks with highly predominant M-links on reducing and∆G links on non-reducing ends. In the LMW fraction, there were almost no G-blocks, with M- and

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G-links on reducing and ∆M and ∆G links on non-reducing ends. This effect was combined with ahigh percentage of oligosaccharides with DP 2 (Supplementary Figure S5).

Figure 4. 1H NMR analysis of products after action of ALFA3 on guluronate-enriched substrate.(A) mannuronate-enriched sodium alginate, (B) total products after action of ALFA3, (C) high-molecularweight fractions after action of ALFA3, and (D) low-molecular weight fractions after action of ALFA3.

The products of ALFA3 and ALFA4 enzymes and the products of their combined action arequite different for the mannuronate-enriched substrate (M:G~3:1). For ALFA3 predominantly H4 ∆Msignals (5.87 and 5.95 ppm) above H4 ∆G (5.76 and 5.80 ppm) are observed (Figure 5A), while ALFA4produces only ∆M signals, predominantly dimers (Figure 5B). The combined action of ALFA3 andALFA4 produces almost exclusively ∆M signals, but the share of dimers decreases (Figure 5C). Thiseffect can be explained by ALFA4 producing fragments that are resistant to the ALFA3 attack.

Figure 5. 1H NMR analysis of action products of ALFA3 (A) and ALFA4 (B) on mannuronate-enrichedsubstrate, as well as products of their joint action (C). M—sodium polymannuronate,MG—mannuronate-enriched sodium alginate, G—guluronate-enriched sodium alginate.

Classification of alginate lyases into M-specific (EC 4.2.2.3), G-specific (EC 4.2.2.11), and alginateoligosaccharide lyases (4.2.2.26) prompted testing of the ALFA3 and ALFA4 substrate specificity on threedifferent types of substrate (Table 1). Considering that polymannuronate- and mannuronate-enrichedsubstrates were digested faster than the guluronate-enriched substrate, both enzymes were classifiedas M-specific (EC 4.2.2.3).

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In the present study, the alginates and their products did not possess cytotoxic effect againstmelanoma SK-MEL-5, SK-MEL-28, and RPMI-7951 cell lines at concentrations up to 800 µg/mL.

Colony formation and growth of SK-MEL-5, SK-MEL-28, and RPMI-7951 cells was tested in thepresence of non-toxic concentrations (200 µg/mL) of alginates and their derivatives using soft agar. Theresults showed that alginates and their products inhibited colony formation of these cells by less than15%, although RPMI-7951 cells were more sensitive to the investigated compounds. Polymannuronateand products of its digestion by ALFA3 (i.e., mannuronate oligosaccharides) demonstrated higheractivity and decreased the number of melanoma RPMI-7951 cells colonies by 20% and 37%, respectively(Figure 6).

Figure 6. The effect of alginates and products of their digestion on colony formation of human cancercells. All concentrations are 200 µg/mL.

Alginates and the products of polymannuronate, mannuronate-enriched and guluronate-enrichedsubstrates digestion by ALFA3 (25–1000 µg/mL) did not affect the development rates and survivabilityof fertilized eggs of the sea urchin Strongylocentrotus intermedius (Figure S6). Both treated and controlembryos reached pluteus stage in 36 h and stayed alive for up to 9–10 days. There was no significantdifference in the morphology of treated and control embryos.

3. Discussion

Two recombinant alginate lyases (ALFA3 and ALFA4) were cloned from the genome of marinebacterium F. algae KMM 3553T. Our results with different digestible substrates determined that ALFA3is a bifunctional endolytic enzyme, while ALFA4 is an M-specific endolytic enzyme. Slight differencesin substrate specificity allowed us to classify ALFA3 and ALFA4 as EC 4.2.2.3 (mannuronate-specificalginate lyase). These bifunctional alginate lyases form a sizable group in the pool of alginolyticenzymes. While there is no specific EC number assigned to this group, these enzymes can be classifiedas either mannuronate-specific (EC 4.2.2.3) or guluronate-specific (EC 4.2.2.11). Previously four alginatelyases from marine bacterium Vibrios plendidus 12B01 were isolated, and two of them could cleave thebond between M and G subunits, while the other two were guluronate-specific [20]. Recently, anotherbifunctional alginate lyase was isolated from marine bacterium Flammeovirga sp. strain MY04 [26]suggesting that the bifunctional mode of action in alginate-utilizing mechanism provided an obviousadvantage for bacteria that could utilize a rich and reliable source of energy.

The relatively low molecular weight of these enzymes makes its recombinant production reliableand efficient due to fewer resources required for their synthesis.

Alginic acids present up to 40% of brown algae dry weight with M/G ratio ranging from 0.21 inSargassum filipendula to 2.28 in Saccharina japonica [27], so M-specific alginate lyase that can degradeeven G-enriched substrate, provides its producer with a convenient tool for survival and adaptation.

Significantly, the optimum pH of ALFA3 was rather sharp for pure M- and G-enriched substrate,but it was much wider (6 to 9) for MG-substrate. This phenomenon can be explained by thedifferences in optimum conditions for digestion of MM and MG bonds, but this hypothesis requiresfurther investigations.

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Analyzing final digestion products of different substrates by ALFA4, we realized thatthe enzyme produced alginate oligosaccharides predominately from mannuronate-enriched andguluronate-enriched substrates but not from pure polymannuronate. Considering that ALFA4 has thehighest activity with mannuronate-enriched substrate, a lower one with pure polymannuronate andrelatively insignificant with guluronate-enriched one, it is possible to hypothesize that ALFA4 cleavesMM-bonds but needs some G-links for more effective substrate binding.

The initial substrates and products of their enzymatic digestion showed only moderate toxicityon human melanoma cells and almost no toxicity on fertilized eggs of sea urchin.

There was no difference if the substances were applied on fresh-fertilized eggs or blastulas (12 hafter fertilization). In some cases, the cells or blastulas survived for 6–7 days longer than controls aftertreatment with products of alginate enzymatic digestion (both HMW and LMW in concentrations0.5-1.0 mg/mL); this effect may be explained by the use of the alginates as a source of energy. If thisis confirmed, the products of enzymatic digestion may be characterized as potentially non-toxic forhealthy cells, opening the possibility for further long-term bioassays with healthy human cells.

In F. algae genome, all alginate lyases are in different gene clusters. This distribution suggests thatalgal alginic acids play a facultative role in the nutrition of this bacterium.

In conclusion, in all cases of alginate substrate digestion with recombinant alginate lyases ALFA3and ALFA4 of marine bacterium F. algae, there is a wide range of alginate oligosaccharides with DPfrom 1 to 20. This observation can be used for screening of products’ biological activity.

4. Materials and Methods

4.1. Genomic DNA Isolation and Construction of Expression Vector

F. algae KMM3553T strain was grown as described by Ivanova et al. [16]. Its genome sequence(GenBank number PRJNA299442) was analyzed for the presence of alginate lyase genes using theRAST server [28]. The strain was obtained from Collection of Marine Microorganisms (KMM) inthe G. B. Elyakov Pacific Institute of Bioorganic Chemistry (PIBOC), Vladivostok, Russia. GenomicDNA was isolated with AxyPrep™Multisource Genomic DNA Purification Miniprep Kit (Axygen,New York, NY, USA) according to the manufacturer’s recommendations. The primers were designedusing restriction-free cloning service [29] based on pColdII plasmid (Takara Bio, Kusatsu, Japan). Theprimers were designed:

ALFA3_dir 5’-CCGAGAACCTTTACTTCCAGGGGGAAGTTACAGATGATAATGCCGAC-3’ALFA3_rev 5’-TCTTAGATTCTGTGCTTTTAAGCAGAGATTACCTATTATAAACTATGTTCGGTTTTTACCTTAT-3’ALFA4_dir 5’- CCGAGAACCTTTACTTCCAGGGGGAAGATCCAGACGACATAGAAGAC-3’ALFA4_rev 5’-TCTTAGATTCTGTGCTTTTAAGCAGAGATTACCTATTACTTTAAGTCTGCCGCCAA-3’

The PCR was done using Phusion HF polymerase (New England Biolabs, Ipswich, MA, USA).Thirty-five cycles of amplification were done to obtain the necessary amount of insert and vector. PCRreaction was controlled by electrophoresis in agarose gel. Sequence analysis was carried out usingthe NCBI BLAST [30]. Domain architectures were identified by InterProScan (http://www.ebi.ac.uk/

interpro/) [31]. Multiple sequence alignment was done with the Clustal Omega server [32]. Competentcells were prepared using Mix & Go E. coli Transformation Kit & Buffer Set (Zymo Research, Irvine, CA,USA). Plasmids were cloned in E. coli XL10 Gold cells (Stratagene, San Diego, CA, USA) and extractedwith QuantumPrep®Plasmid Miniprep Kit (Bio-Rad, Hercules, CA, USA).

4.2. Expression and Purification of Recombinant ALFA3 and ALFA4

E. coli Arctic Express (DE3) (Agilent, Santa Clara, CA, USA) cells with plasmids pColdII-ALFA3and pColdII-ALFA4 were grown overnight at 37 ◦C on LB plates containing 100 µg/mL ampicillin. Asingle colony was picked and grown at 200 rpm in LB with 100 µg/mL ampicillin at 37 ◦C for 12 h,

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then transferred to fresh LB with 100 µg/mL ampicillin. Cells were grown up to an optical density ofapproximately 0.6 OD600; expression was induced by IPTG (0.2 mM) and carried out for 16 h at 16 ◦C.Recombinant cells were collected by centrifugation at 6000 relative centrifugal forces (rcf) for 20 min at4 ◦C, resuspended in 20 mM phosphate buffer (pH 7.4), disrupted by sonication, and centrifuged at6000 rcf during 20 min at 4 ◦C. The resulting supernatant was applied to a 2 mL column of Ni-NTAagarose (General Electric, Chicago, IL, USA) pre-equilibrated with 10 mM sodium phosphate buffer(pH 7.4) with 0.5 M NaCl and 20 mM imidazole. Chromatography was performed with a lineargradient of 20–500 mM imidazole (total volume 40 mL), the flow rate of 0.5 mL/min. His-tag wasremoved by TEV-protease (Sigma-Aldrich, St. Louis, MO, USA). The enzyme was re-equilibrated insuccinate buffer pH 6.0. This method produced pure alginate lyases ALFA3 and ALFA4 (single bandby SDS-PAGE, see Figure S7).

4.3. Analytical Methods

Protein concentration was measured by the Bradford procedure with bovine serum albumin (BSA)as a standard [33]. The homogeneity and molecular weights of the enzymes were determined bySDS PAGE using the method of Laemmli [34] and 12% acrylamide gels. The Precision Plus Protein™Standards (Bio-Rad, Hercules, CA, USA) was used as standard.

4.4. Electrophoresis of Carbohydrates (C-PAGE)

The substrates and products of their hydrolysis (15 µL, 4 mg/mL) were mixed with 5 µL of loadingbuffer containing a 20% solution of glycerol in water and 0.02% of phenol red. The samples (10 µL)were separated by electrophoresis through a 5% (w/v) stacking gel with 50 mM Tris–HCl buffer pH 6.8and 27% (w/v) resolving polyacrylamide gel with 150 mM Tris–HCl buffer pH 8.8. The gel was 1 mmthick. The gel was stained with 0.02% O-toluidine blue (Sigma-Aldrich, St. Louis, MO, USA) and 0.3%alcian blue in EtOH, AcOH, and H2O with a volume ratio of 2:1:1. The gel images were visualizedusing a calibrated densitometer DS-800 (Bio-Rad).

4.5. Substrates and Enzyme Assay

Sodium polymannuronate was isolated from brown alga Saccharina cichorioides in the Laboratoryof Enzyme Chemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Russia PIBOC FEBRAS. Guluronate-enriched sodium alginate (BDH, UK, M:G~1:2), and mannuronate-enriched sodiumalginate (Sigma-Aldrich, St. Louis, MO, USA, M:G~3:1) were also used.

The activity of alginate lyase was deduced from the increase in reducing sugars [35]. The incubatedmixture contained 100 µL of the enzyme, 200 µL of sodium alginate solution (4 mg/mL in 25 mMsuccinic buffer, рН6.0). The incubation was for 1 h at 37 ◦C to cleave approximately 10% of thesubstrate. Unit of activity is determined as the amount of the enzyme that catalyzed the formation of1 µmole of monomer per min. Enzyme and substrate controls were used to exclude false positives. Allexperiments were done at least three times.

4.6. Enzyme Characterization

The optimum pH values for ALFA3 and ALFA4 were determined by the standard activity assayfor 1 h at 37 ◦C using 0.2 M citrate-phosphate buffer in the range of pH 2.0–10.0. The reaction mixturecontained 100 µL of the enzyme solution, 200 µL of the substrate solution (4 mg/mL), and 300 µL of abuffer with various pH.

The optimum temperature was determined by the standard activity assay for 1 h at temperaturesbetween 25 and 55 ◦C with 5 ◦C intervals and the concentration of reducing sugars was measured.

Thermal stability was estimated by measuring the residual activity by standard assay afterpre-incubation of the enzyme at temperatures between 10 and 70 ◦C for 1 h.

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Km was calculated from the Lineweaver-Burk plot using data collected by measuring the rateof mannuronate-enriched sodium alginate hydrolysis under the standard assay conditions for 1 h at37 ◦C from 0.025 to 2.0 mg/mL.

4.7. Enzyme Classification

Classification of the enzymes by amino acid sequences was done using InterPro [36]. Classificationof the enzymes by the type of activity was done according to the enzyme activity on differentsubstrates: polymannuronate, mannuronate-enriched alginate, and guluronate-enriched alginate (seethe Section 4.5).

4.8. Obtaining the Products of Exhaustive Hydrolysis by ALFA3 and ALFA4

For exhaustive hydrolysis, 100 µL of enzyme solution (1 mg/mL) was added to a solution ofdifferent sodium alginate substrates (4 mg/mL, 25 mL), and the mixture was incubated at 37 ◦C.Aliquots (50 µL) were taken at 0, 0.5, 1.5, 3.5, 6, 24, 48, 72, and 96 h, and the reaction was terminatedby heating in a solid-state thermostat (DNA Technology, Russia) for 1 min at 99 ◦C. Products wereanalyzed by C-PAGE. After 96 h, the reaction mixture was concentrated by rotary evaporator (IKA,Staufen, Germany) at 50 ◦C and lyophilized.

Fractionation of alginate oligosaccharides was performed with ethanol precipitation. After theend of reaction, the enzyme was inactivated by heating the reaction mixture at 90 ◦C during 1 hand then cooling it to the room temperature. Ninety-five percent ethanol was added to aliquots ofinactivated reaction mixture in ration (ethanol:mixture) from 0.1:1 to 0.9:1, so the final concentrationsof ethanol were from 8.6 to 45%. The aliquots were stirred well and then incubated at 4 ◦C for 18 h.After incubation, the aliquots were centrifuged at 9000 rcf during 30 min, and the supernatants werecollected, concentrated by rotary evaporator (IKA, Germany) at 50 ◦C, lyophilized, resuspended inwater (up to 4 mg/mL), and analyzed by C-PAGE.

1H NMR spectra were recorded using a Bruker DRX 500 spectrometer 500 MHz (Bruker, Germany).There were taken the spectra of the native substrates and the reaction mixtures (96h of reaction) (allsamples by 10 mg). The reactions of hydrolysis (4 mg/mL of substrate, 0.1 U/mL of enzyme) were runin 25 mM succinic buffer pH 6.0 at 37 ◦C, so after resuspension, pH was close to 6.0. The products ofthe exhaustive hydrolysis were freeze-dried, resuspended in D2O, and the spectra were taken at 35 ◦C.The volume of the sample was 0.5 mL.

4.9. Bioassays with Human Melanoma Cells

4.9.1. Chemicals and Cell Lines

Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline (PBS), l-glutamine,penicillin-streptomycin solution, trypsin, fetal bovine serum (FBS), sodium hydrocarbonate (NaHCO3),and agar were purchased from Biolot (Moscow, Russia).

Human melanoma SK-MEL-5 (ATCC®no. HTB-70™), SK-MEL-28 (ATCC®no.HTB-72™),RPMI-7951 (ATCC®no. HTB-66™) cell lines were purchased from the American Type CultureCollection (ATCC, Manassas, VA, USA).

4.9.2. Cells Cultivation

Human melanoma SK-MEL-5, SK-MEL-28, and RPMI-7951 cell lines were cultured inDMEM medium, supplemented with 10% fetal bovine serum (FBS), 200 mM l-glutamine, andpenicillin-streptomycin solution. The cell cultures were maintained at 37 ◦C in a humidified atmospherecontaining 5% CO2.

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4.9.3. Cytotoxicity Assay

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay was used as the indicator of cell viability determined by mitochondrial-dependentreduction of formazan. In brief, cells were seeded at a density of 1.0 × 104 cells per well in 200 µL ofthe complete medium in 96-well plates. After 24 h incubation, the attached cells were treated withvarious concentrations of alginates and its products (100, 200, 400, 800 µg/mL), while the control wasmock-treated with the DMEM medium only. Cells were cultured for an additional 24 h at 37 ◦C in 5%CO2 incubator. After incubation MTS reagent (20 µL) was added to each well, and cells were incubatedfor 3 h at 37 ◦C in 5% CO2. Absorbance was measured at 490/630 nm by a microplate reader PowerWave XS (Bio-Tek, Winooski, VT, USA). All tests were carried out in triplicate.

4.9.4. Anchorage-Independent Colony Formation Assay

SK-MEL-5 (A), SK-MEL-28 (B), and RPMI-7951 cells (2.4 × 104/mL) were seeded in six-well platewith or without alginates and its products (200 µg/mL) in 1 mL of 0.3% Basal Medium Eagle (BME)agar containing 10% FBS, 2 mM l-glutamine, and 25 µg/mL gentamicin. The cultures were maintainedat 37 ◦C in a 5% CO2 incubator for 14 days, and the cell’s colonies were scored using a microscopeMotic AE 20 (Motic, Xiamen, China) and the Motic Image Plus computer program.

4.10. Bioassays with Sea Urchin Embryos

Sea urchins Strongylocentrotus intermedius were collected in Troitsa Bay, Sea of Japan, Russia, inSeptember 2019 as described [37]. In brief, the eggs and developing embryos of the sea urchin wereused at a concentration of 2500 cells per mL of seawater. The concentrations of substances were between25 and 1000 µg/mL. The substances were applied to fertilized eggs at the zygote stage and activelymobile embryos at the blastula stage. The development was observed with Motic AE21 microscopeand Levenhuk C-series camera (Levenhuk, Tampa, FL, USA).

4.11. Statistical Assay

All assays were performed at least in triplicate. The results are expressed as the mean ± standarddeviation (SD). A Student’s t-test was used to evaluate the data with the following significance levels:* p < 0.05, ** p < 0.01, *** p < 0.001.

Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/18/2/130/s1.

Author Contributions: Molecular biology and protein expression, A.B. and A.S.; enzymatic transformationof alginate, A.B.; chemical properties of enzymes and alginate oligosaccharides, A.B.; NMR, A.R.; bioassayson human melanoma cells, O.M and S.E.; bioassays on sea urchin embryos, A.B. and M.K.; writing—originaldraft preparation, A.B.; writing—review and editing, A.B., M.K. and S.E.; project administration, M.K.; fundingacquisition, S.E. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by RFBR, grant number 18-04-00905_a.

Acknowledgments: The study was carried out on the equipment of the Collective Facilities Center “The FarEastern Center for Structural Molecular Research (NMR/MS) PIBOC FEB RAS”.

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Ertesvag, H. Alginate-modifying enzymes: biological roles and biotechnological uses. Front. Microbiol. 2015,6, 523.

2. Wong, T.Y.; Preston, L.A.; Schiller, N.L. ALGINATE LYASE: Review of major sources and enzymecharacteristics, structure-function analysis, biological roles, and applications. Annul. Rev. Microbiol.2000, 54, 289–340. [CrossRef] [PubMed]

3. Farrell, E.K.; Tipton, P.A. Functional characterization of AlgL, an alginate lyase from Pseudomonas aeruginosa.Biochemistry 2012, 51, 10259–10266. [CrossRef] [PubMed]

Mar. Drugs 2020, 18, 130 11 of 12

4. Zhu, B.; Ning, L.; Jiang, Y.; Ge, L. Biochemical Characterization and Degradation Pattern of a Novel Endo-TypeBifunctional Alginate Lyase AlyA from Marine Bacterium Isoptericola halotolerans. Mar. Drugs 2018, 16,258. [CrossRef] [PubMed]

5. Garron, M.L.; Cygler, M. Structural and mechanistic classification of uronic acid-containing polysaccharidelyases. Glycobiology 2010, 20, 1547–1573. [CrossRef] [PubMed]

6. Lombard, V.; Bernard, T.; Rancurel, C.; Brumer, H.; Coutinho, P.M.; Henrissat, B. A hierarchical classificationof polysaccharide lyases for glycogenomics. Biochem. J. 2010, 432, 437–444. [CrossRef] [PubMed]

7. Yamasaki, M.; Ogura, K.; Hashimoto, W.; Mikami, B.; Murata, K. A structural basis for depolymerization ofalginate by polysaccharide lyase family-7. J. Mol. Biol. 2005, 352, 11–21. [CrossRef]

8. Garron, M.L.; Cygler, M. Uronic polysaccharide degrading enzymes. Curr. Opin. Struct. Biol. 2014, 28, 87–95.[CrossRef]

9. Iwamoto, M.; Kurachi, M.; Nakashima, T.; Kim, D.; Yamaguchi, K.; Oda, T.; Iwamoto, Y.; Muramatsu, T.Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production fromRAW264.7 cells. Febs Lett. 2005, 579, 4423–4429. [CrossRef]

10. Yamamoto, Y.; Kurachi, M.; Yamaguchi, K.; Oda, T. Stimulation of multiple cytokine production in mice byalginate oligosaccharides following intraperitoneal administration. Carbohydr. Res. 2007, 342, 1133–1137.[CrossRef]

11. Chen, J.; Hu, Y.; Zhang, L.; Wang, Y.; Wang, S.; Zhang, Y.; Guo, H.; Ji, D.; Wang, Y. Alginate OligosaccharideDP5 Exhibits Antitumor Effects in Osteosarcoma Patients following Surgery. Front. Pharmacol. 2017, 8, 623.[CrossRef] [PubMed]

12. Falkeborg, M.; Cheong, L.Z.; Gianfico, C.; Sztukiel, K.M.; Kristensen, K.; Glasius, M.; Xu, X.; Guo, Z. Alginateoligosaccharides: Enzymatic preparation and antioxidant property evaluation. Food Chem. 2014, 164, 185–194.[CrossRef] [PubMed]

13. Li, S.Y.; He, N.N.; Wang, L.N. Efficiently Anti-Obesity Effects of Unsaturated Alginate Oligosaccharides(UAOS) in High-Fat Diet (HFD)-Fed Mice. Mar. Drugs 2019, 17, 540. [CrossRef] [PubMed]

14. Sandberg, A.S.; Andersson, H.; Bosaeus, I.; Carlsson, N.G.; Hasselblad, K.; Harrod, M. Alginate, small bowelsterol excretion, and absorption of nutrients in ileostomy subjects. Am. J. Clin. Nutr. 1994, 60, 751–756.[CrossRef] [PubMed]

15. Uno, T.; Hattori, M.; Yoshida, T. Oral administration of alginic acid oligosaccharide suppresses IgE productionand inhibits the induction of oral tolerance. Biosci. Biotechnol. Biochem. 2006, 70, 3054–3057. [CrossRef]

16. Ivanova, E.P.; Alexeeva, Y.V.; Flavier, S.; Wright, J.P.; Zhukova, N.V.; Gorshkova, N.M.; Mikhailov, V.V.;Nicolau, D.V.; Christen, R. Formosa algae gen. nov., sp. nov., a novel member of the family Flavobacteriaceae.Int. J. Syst. Evol. Microbiol. 2004, 54, 705–711. [CrossRef]

17. Silchenko, A.S.; Ustyuzhanina, N.E.; Kusaykin, M.I.; Krylov, V.B.; Shashkov, A.S.; Dmitrenok, A.S.;Usoltseva, R.V.; Zueva, A.O.; Nifantiev, N.E.; Zvyagintseva, T.N. Expression and biochemical characterizationand substrate specificity of the fucoidanase from Formosa algae. Glycobiology 2017, 27, 254–263. [CrossRef]

18. Silchenko, A.S.; Rasin, A.B.; Kusaykin, M.I.; Kalinovsky, A.I.; Miansong, Z.; Changheng, L.; Malyarenko, O.;Zueva, A.O.; Zvyagintseva, T.N.; Ermakova, S.P. Structure, enzymatic transformation, anticancer activity offucoidan and sulphated fucooligosaccharides from Sargassum horneri. Carbohydr. Polym. 2017, 175, 654–660.[CrossRef]

19. Kusaykin, M.I.; Belik, A.A.; Kovalchuk, S.N.; Dmitrenok, P.S.; Rasskazov, V.A.; Isakov, V.V.; Zvyagintseva, T.N.A new recombinant endo-1,3-beta-D-glucanase from the marine bacterium Formosa algae KMM 3553: enzymecharacteristics and transglycosylation products analysis. World J. Microbiol. Biotechnol. 2017, 33, 12. [CrossRef]

20. Badur, A.H.; Jagtap, S.S.; Yalamanchili, G.; Lee, J.K.; Zhao, H.; Rao, C.V. Alginate Lyases fromAlginate-Degrading Vibrio splendidus 12B01 Are Endolytic. Appl. Environ. Microbiol. 2015, 81, 1856–1864.[CrossRef]

21. Boucelkha, A.; Petit, E.; Elboutachfaiti, R.; Molinie, R.; Amari, S.; Yahaoui, R. Production of guluronateoligosaccharide of alginate from brown algae Stypocaulon scoparium using an alginate lyase. J. Appl. Phycol.2017, 29, 509–519. [CrossRef]

22. Campa, C.; Holtan, S.; Nilsen, N.; Bjerkan, T.M.; Stokke, B.T.; Skjak-Braek, G. Biochemical analysis of theprocessive mechanism for epimerization of alginate by mannuronan C-5 epimerase AlgE4. Biochem. J. 2004,381, 155–164. [CrossRef] [PubMed]

Mar. Drugs 2020, 18, 130 12 of 12

23. Fertah, M.; Belfkira, A.; Dahmane, E.M.; Taourirte, M.; Brouillette, F. Extraction and characterization ofsodium alginates from Moroccan Laminaria digitata brown seaweed: Sodium alginates from Laminariadigitata. Arabian J. Chem. 2017, 10, S3707–S3714. [CrossRef]

24. Santi, C.; Coppetta, D.; Santoro, S.; Basta, G.; Montanucci, P.; Racanicchi, L.; Calafiore, R. NMR Analysis ofNon Hydrolyzed Samples of Sodium Alginate. In Proceedings of the 12th International Electronic Conferenceon Synthetic Organic Chemistry (ECSOC’12), Basel, Switzerland, 1–30 November 2008.

25. Thomas, F.; Lundqvist, L.C.E.; Jam, M.; Jeudy, A.; Barbeyron, T.; Sandstrom, C.; Michel, G.; Czjzek, M.Comparative Characterization of Two Marine Alginate Lyases from Zobellia galactanivorans Reveals DistinctModes of Action and Exquisite Adaptation to Their Natural Substrate. J. Biol. Chem. 2013, 288, 23021–23037.[CrossRef] [PubMed]

26. Peng, Y.; Liu, G.L.; Yu, X.J.; Wang, X.H.; Jing, L.; Chi, Z.M. Cloning of exo-beta-1,3-glucanase gene from amarine yeast Williopsis saturnus and its overexpression in Yarrowia lipolytica. Mar. Biotechnol. (NY) 2011,13, 193–204. [CrossRef] [PubMed]

27. Belik, A.A.; Silchenko, A.S.; Kusaykin, M.I.; Zvyagintseva, T.N.; Ermakova, S.P. Alginate Lyases: Substrates,Structure, Properties, and Prospects of Application. Russian J. Bioorg. Chem. 2018, 44, 386–396. [CrossRef]

28. Aziz, R.K.; Bartels, D.; Best, A.A.; DeJongh, M.; Disz, T.; Edwards, R.A.; Formsma, K.; Gerdes, S.; Glass, E.M.;Kubal, M.; et al. The RAST Server: rapid annotations using subsystems technology. BMC Genomics 2008, 9,75. [CrossRef]

29. Van den Ent, F.; Löwe, J. RF cloning: A restriction-free method for inserting target genes into plasmids.J. Biochem. Biophy. Methods 2006, 67, 67–74. [CrossRef]

30. Johnson, M.; Zaretskaya, I.; Raytselis, Y.; Merezhuk, Y.; McGinnis, S.; Madden, T.L. NCBI BLAST: A betterweb interface. Nucleic Acids Res. 2008, 36, W5–W9. [CrossRef]

31. Jones, P.; Binns, D.; Chang, H.-Y.; Fraser, M.; Li, W.; McAnulla, C.; McWilliam, H.; Maslen, J.; Mitchell, A.;Nuka, G.; et al. InterProScan 5: Genome-scale protein function classification. Bioinformatics 2014, 30,1236–1240. [CrossRef]

32. Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T.J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.;Söding, J.; et al. Fast, scalable generation of high-quality protein multiple sequence alignments using ClustalOmega. Mol. Sys. Biol. 2011, 7, 539. [CrossRef]

33. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizingthe principle of protein-dye binding. Analy. Biochem. 1976, 72, 248–254. [CrossRef]

34. Laemmli, U.K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature1970, 227, 680–685. [CrossRef]

35. Nelson, N. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem.1944, 153, 375–380.

36. Mitchell, A.L.; Attwood, T.K.; Babbitt, P.C.; Blum, M.; Bork, P.; Bridge, A.; Brown, S.D.; Chang, H.Y.;El-Gebali, S.; Fraser, M.I.; et al. InterPro in 2019: improving coverage, classification and access to proteinsequence annotations. Nucleic Acids Res. 2019, 47, D351–D360. [CrossRef] [PubMed]

37. Kiseleva, M.I.; Balabanova, L.A.; Rasskazov, V.A.; Zvyagintseva, T.N. Effect of 1,3;1,6-beta-D-glucans ondeveloping sea urchin embryos. Mar. Biotechnol. 2008, 10, 466–470. [CrossRef]

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